Universal carrier for targeting molecules to GB3 receptor expressing cells

Abstract

The present invention concerns an universal polypeptidic carrier for targeting directly or indirectly a molecule to Gb3 receptor expressing cells and having the following formula STxB-Z(n)-Cys, wherein: STxB is the Shiga Toxin B subunit or a functional equivalent thereof, Z is an amino-acid devoided of sulfydryl group, n being 0, 1 or a polypeptide, Cys is the amino-acid Cysteine, and the use thereof for MHC class I and MHC class II presentation of antigens.

The invention claimed is: 
     
       1. An isolated polynucleotide selected from the group consisting of:
 a) a polynucleotide comprising the nucleotide sequence STxB encoding the Shiga Toxin B subunit or a functional equivalent thereof bearing at its 3′ end the codon TGT, or the codon TGC encoding cysteine, wherein the functional equivalent thereof binds to the Gb3 receptor and triggers the internalization of an antigen and its presentation in an MHC class I and MHC class II restricted pathway on the same antigen presenting cell; 
 b) a polynucleotide comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence encoding the Shiga Toxin B subunit or a functional equivalent thereof, bearing at its 3′ end the codon TGT or TGC, wherein the functional equivalent thereof binds to the Gb3 receptor and triggers the internalization of an antigen and its presentation in an MHC class I and MHC class II restricted pathway on the same antigen presenting cell and 
 c) a nucleotide sequence complementary to the sequence in a) or b). 
 
     
     
       2. The polynucleotide according to  claim 1 , comprising SEQ ID No. 2. 
     
     
       3. A recombinant vector or plasmid, comprising a polynucleotide sequence according to  claim 1 . 
     
     
       4. A recombinant cell line obtained by transformation with the recombinant vector according to  claim 3 . 
     
     
       5. The recombinant cell line according to  claim 4 , which is a prokaryotic cell line. 
     
     
       6. The recombinant cell line according to  claim 5 , which prokaryotic cell line is  E. coli.    
     
     
       7. The recombinant cell line according to  claim 6 , deposited at the CNCM on Dec. 19, 2000 under accession number I-2604. 
     
     
       8. A method for constructing a recombinant vector according to  claim 3  comprising:
 a) providing a plasmid comprising a STxB sequence; 
 b) applying two PCR amplification steps using two couples of primers AA′ and BB′ wherein A and B are complementary to each other and comprise the Cys codon and A′ and B′ are outside the STxB sequence; 
 c) isolating the amplified fragments; 
 d) hybridizing the amplified fragments; 
 e) applying a PCR amplification on the hybridized fragments; and 
 f) inserting the amplified fragments into a plasmid. 
 
     
     
       9. The method according to  claim 8 , wherein in step f) the fragments are inserted into a SphI and SalI restriction site of the plasmid pSU108. 
     
     
       10. A process for producing an isolated polypeptide comprising:
 a). culturing a recombinant cell line of  claim 4 ; 
 b) obtaining a periplasmic extract of said cells; and 
 c) purifying said polypeptide. 
 
     
     
       11. The process according to  claim 10  wherein in step a) the cell line is  E. coli  and in step c) the purification is by anion exchange column chromatography followed by a gel filtration column chromatography.